Liquid chromatography (LC) columns have been extensively developed and are used routinely in both analytical and preparative chromatography. The separation in a chromatography column of a sample (also termed an analyte or solute) comprising a mixture of components is achieved by dissolving the sample in a liquid mobile phase and passing the mobile phase through a stationary phase typically packed within a tubular column, thereby causing the sample to separate into its components due to different partitioning between the mobile and stationary phases of the different components (i.e. the components have different partition coefficients). In liquid chromatography the stationary phase is typically in the form of a bed of particles packed within the column. This invention relates to such so-called packed columns.
Silica particles are commonly used as the stationary phase bed. Non-porous silica particles have a low sample capacity. Therefore, porous silica particles are commonly used which contain a network of pores to increase the surface area of the stationary phase and thus improve the capacity of the separation. The pores may be micro pores of less than 2 nm size, meso pores of 2 nm to 50 nm, or macro pores of greater than 50 nm. There has been a trend to reduce the size of the porous particles to improve the kinetics and resolution of the separation but this has been at the expense of increasing operating pressure. Porous particles can also be of excessive particle size distribution, which limits peak resolution. An alternative to fully porous silica particles in which the pores extend throughout the bulk of the particles has been to use so-called superficially porous particles, i.e. particles which are porous only at their surface. These enable a reasonable operating pressure to be used but still with a high resolution. They also offer the possibility of narrow particle size distributions. Superficially porous particles comprise a non-porous core having an outer porous shell. The porous shell results in short mass transfer distances and hence fast mass transfer and fast separation of samples. This is important for separation of large biomolecules such as proteins for example. Superficially porous particles are available commercially for HPLC in Poroshell™ columns from Agilent and Accucore™ columns from Thermo Scientific.
In a known procedure described in U.S. Pat. No. 3,505,785, superficially porous silica particles are prepared via multilayer coatings of silica colloids on the surface of non-porous silica microspheres acting as solid cores. Between two and thirty layers of colloid particles are described. This arrangement is induced by surface compatibility and in some cases surface pretreatment or modification of silica particles is necessary to induce interactions. This procedure is rather complex as the actual core and shell particles are prepared individually, followed by the core coating step. This procedure is also a lengthy process as each layer must be applied in a separate step and it is difficult to reproduce the final superficially porous particle size and size distribution.
Another method of preparing superficially porous silica is a coacervation method, described in J. J. Kirkland, F. A. Truszkowski, and C. H. Dilks Jr, G. S. Engel, Journal of Chromatography A, 890 (2000) 3-13, in which solid (i.e. non-porous) silica microspheres are coated by a coacervate of a polymer and silica sol, with the polymer being subsequently removed by heating at high temperature. However, this multi-stage technique has further disadvantages in that some core particles may not be coated leaving non-porous particles and some fully porous particles may be formed. Similarly, WO 2012/018598, published after the priority date of the present application, describes superficially porous particles formed by at least a two-step synthesis, wherein seeded silica particles or other particles were synthesized first and then the porous layers were produced on top of that.
The above techniques employ a porous layer on the surface of silica microparticles as the basis for superficially porous particles. Another method, described in US 2010/0051877 A, involves the pseudomorphic transformation of the surface of silica microparticles. During the process, the outer layer of the core particle is dissolved and re-precipitates to form a porous layer on the surface. However, the technique requires the core particles to be formed in a first reaction and then recovered in order to be processed in a further reaction to effect the pseudomorphic transformation of the surface to form the superficially porous particles. Thus, the technique has disadvantages in terms of excessive preparation time and reproducibility. The cores must be grown first, checked for quality control and then classified, which can take weeks. Then the shell has to be developed onto the core and grown to a given thickness. In so doing, the particle size changes and the particle size distribution broadens, possibly requiring further classification, which again may take weeks.
Conventionally, silica microparticles themselves are made using variations of the Stöber process (W. Stöber, A. Fink, E. Bohn, Journal of Colloid and Interface Science, Volume 26, Issue 1, 1968, p. 62-69) in which tetraethyl orthosilicate (TEOS) is added to an excess of water containing an alcohol such as ethanol and ammonia. Hydrolysis and condensation of the TEOS produces silica particles.
The use of mercapto-silanes as a precursor silica source instead of TEOS to produce porous, monodisperse silica microparticles has been described previously in Lee et al, Langmuir, 2007, 23 (22), pp 10875-10878; Lee et al, Microporous and Mesoporous Materials, 2001, 50 pp 77-90 and Lu et al, Langmuir, 2011, 27 (7), pp 3372-3380. Generally, it was found that increasing the concentration of the mercapto-silane increased the particle size. Increasing the base concentration on the other hand decreased the particle size. The silica microparticles reported in those studies are porous and mostly smooth, i.e. without any reported superficial porosity.
Against this background the present invention has been made.